The method used for the isolation of large scale cosmid and plasmid DNA is an
unpublished modification (16) of an alkaline lysis procedure (17,18) followed
by equilibrium ultracentrifugation in cesium chloride-ethidium bromide
gradients (1). Briefly, cells containing the desired plasmid or cosmid are
harvested by centrifugation, incubated in a lysozyme buffer, and treated with
alkaline detergent. Detergent solubilized proteins and membranes are
precipitated with sodium acetate, and the lysate is cleared first by filtration
of precipitate through cheesecloth and then by centrifugation. The
DNA-containing supernatant is transferred to a new tube, and the plasmid or
cosmid DNA is precipitated by the addition of polyethylene glycol and collected
by centrifugation. The DNA pellet is resuspended in a buffer containing cesium
chloride and ethidium bromide, which is loaded into polyallomer tubes and
subjected to ultracentrifugation overnight. The ethidium bromide stained
plasmid or cosmid DNA bands, equilibrated within the cesium chloride density
gradient after ultracentrifugation, are visualized under long wave UV light and
the lower band is removed with a 5 cc syringe. The intercalating ethidium
bromide is separated from the DNA by loading the solution onto an equilibrated
ion exchange column. The A260 containing fractions are pooled, diluted, and
ethanol precipitated, and the final DNA pellet is resuspended in buffer and
assayed by restriction digestion as detected on agarose gel electrophoresis.

During the course of this work several modifications to the above protocol
were made. For example, initially cell growth times included three successive
overnight incubations, beginning with the initial inoculation of 3 ml of
antibiotic containing media with the plasmid or cosmid-containing bacterial
colony, and then increasing the culture volume to 50 ml, and then to 4 l.
However, it was observed that recombinant cosmid DNA isolated from cell
cultures grown under these conditions, in contrast to recombinant plasmid DNA,
was contaminated with deleted cosmid DNA molecules. However, these deletions
are avoided by performing each of the three successive incubations for eight
hours instead of overnight, although a slight yield loss accompanied the
reduced growth times.

Recently, a diatomaceous earth-based (19-22) method was used to isolate the
plasmid or cosmid DNA from a cell lysate. The cell growth, lysis, and cleared
lysate steps are performed as described above, but following DNA precipitation
by polyethylene glycol, the DNA pellet is resuspended in RNase buffer and
treated with RNase A and T1. Nuclease treatment is necessary to remove the RNA
by digestion since RNA competes with the DNA for binding to the diatomaceous
earth. After RNase treatment, the DNA containing supernatant is bound to
diatomaceous earth in a chaotropic buffer of guanidine hydrochloride by
incubation at room temperature. The DNA-associated diatomaceous earth then is
collected by centrifugation, washed several times with ethanol buffer and
acetone, dried, and then resuspended in buffer. The DNA is eluted during
incubation at 65degC, and the DNA-containing supernatant is
collected after centrifugation and separation of the diatomaceous earth
particles. The DNA recovery is measured by taking absorbance readings at 260
nanometers. After concentration by ethanol precipitation, the DNA is assayed
by restriction digestion.

Protocol

1. Pick a colony of bacteria harboring the plasmid or cosmid DNA of interest
into a 12 X 75 mm Falcon tube containing 2 ml of LB media supplemented with the
appropriate antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37deg
C 8-10 hours with shaking at 250 rpm. Transfer the culture
to an Ehrlenmeyer flask containing 50 ml of similar media, and incubate further
for 8-10 hours. Transfer 12.5 ml of the culture to each of 4 liters of similar
media, and incubate for an additional 8-10 hours.

2. Harvest the cells by centrifugation at 7000 rpm for 20 minutes in 500 ml
bottles in the RC5-B using the GS3 rotor. Resuspend the cell pellets in old
media and transfer to two bottles, centrifuge as before, and decant the media.
The cell pellets can be frozen at -70degC at this point.

3. Resuspend the cell pellets in a total of 70 ml of GET/Lysozyme solution (35
ml for each bottle) by gently teasing the pellet with a spatula and incubate
for 10 minutes at room temperature. (Note: Do not vortex the lysate at any
time because this may shear the chromosomal DNA).

4. Add a total of 140 ml of alkaline lysis solution (70 ml for each bottle),
gently mix, and incubate for 5 minutes in an ice-water bath.

5. Add 105 ml of 3M NaOAc, pH 4.8 (52.5 ml for each bottle), cap tightly,
gently mix by inverting the bottle a few times, and incubate in an ice-water
bath for 30-60 minutes.

6. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal
DNA by pouring through a double-layer of cheesecloth. Transfer the lysate into
250 ml centrifuge bottle, centrifuge at 10,000 rpm for 30 minutes at 4deg
C in the RC5-B using the GSA rotor.

For cesium chloride-gradient purification:

7a. Pool the cleared supernatants into to a clean beaker, add one-fourth volume
of 50% PEG/0.5 M NaCl, swirl to mix, and incubate in an ice-water bath for 1-2
hours.

8a. Collect the PEG-precipitated DNA by centrifugation in 250 ml bottles at
7000 rpm for 20 minutes at 4degC in the RC5-B using the GSA
rotor.

11a. Centrifuge at 60,000 rpm to 16-20 hours at 15-20degC in
the Sorvall OTD-75B ultracentrifuge (DuPont) using the T-865 rotor.

12a. Visualize the ethidium bromide stained DNA under long-wave UV light, and
remove the lower DNA band using a 5 cc syringe and a 25 gauge needle. (It may
be helpful first to remove and discard the upper band).

13a. To remove the ethidium bromide, load the DNA sample onto an equilibrated
1.5 ml Dowex column, and collect 0.5 ml fractions. Equilibrate the Dowex AG
resin (BioRad) by successive centrifugation, resuspension, and decanting with
1M NaOH, water, and then 1M Tris-HCl, pH 7.6 until the Dowex solution has a pH
of 7.6.

14a. Pool fractions with an A260 of 1.00 or greater into 35 ml Corex glass
tubes, add one volume of ddH2O, and ethanol precipitate by adding 2.5 volumes
of cold 95% ethanol. Incubate at least 2 hours at -20degC,
centrifuge at 10,000 rpm for 45 minutes in the RC5-B using the SS-34 rotor.
Gently decant the supernatant, add 80% ethanol, centrifuge as before, decant,
and dry the DNA pellet in a vacuum oven.

15a. Resuspend the DNA in 10:0.1 TE buffer.

For diatomaceous earth-based purification:

7b. Pool the supernatants from step 6 into 500 ml bottles and add DNase-free
RNase A and RNase T1 such that the final concentration of RNase A is 40 ug/ml
and RNase T1 is 40 U/ml. Incubate in a 37degC water bath
for 30 minutes.

8b. Add an equal volume of isopropanol and precipitate at room temperature for
5 minutes. Centrifuge at 9,000 rpm for 30 minutes in the RC5-B using the GS3
rotor. Decant the supernatant and drain the DNA pellet.

9b. Resuspend each DNA pellet in 20 ml 10:1 TE buffer, and add 40 ml of
de-fined diatomaceous earth in guanidine-HCl (100 mg/ml) to each bottle. Allow
the DNA to bind at room temperature for 5 minutes with occasional mixing.
Centrifuge at 9,000 for 10 minutes in the RC5-B using the GS3 rotor.

10b. Decant the supernatant, resuspend each pellet in 40 ml of diatomaceous
earth-wash buffer, and centrifuge as above.

11b. Decant the supernatant, resuspend each pellet in 40 ml of acetone, and
centrifuge as above.

12b. Decant the supernatant and dry the pellet in a vacuum oven.

13b. Resuspend the pellet in 20 ml of 10:1 TE buffer, and elute the bound DNA
by incubation at 65degC for 10 minutes with intermittent
mixing.

14b. Remove the diatomaceous earth by centrifugation at 9,000 rpm for 10
minutes in the RC5-B using the GS3 rotor. Repeat if necessary.

A midi-prep double-stranded DNA isolation has been developed to generate a
sufficient amount of template DNA for several Sequenase[TM]
catalyzed fluorescent terminator reactions. Here, one bacterial colony which
harbored the plasmid of interest is picked into 3 ml of liquid media containing
ampicillin and incubated in a 37degC shaker for 8-10 hours.
At this time, the culture is transferred into 50 ml of ampicillin-containing
media and incubated further for 10-12 hours. After harvesting the cells by
centrifugation, a diatomaceous earth-based alkaline-lysis purification method
(19-22) is performed, similar to that discussed above for large scale DNA
isolation. The purified DNA is crudely assayed for concentration and purity by
agarose gel electrophoresis against known standards. The approximate yield of
double-stranded DNA using this method is 1 ug of DNA per ml of cell culture.
For a 50 ml cell culture, about 50 ug of DNA are recovered, and 5 ug are used
typically in a Sequenase[TM] terminator reaction.

Note: This procedure is the method of choice for isolating double
stranded plasmid-based templates for the Sequenase Dye-Labeled Terminator
Sequencing Reactions.

Protocol

1. Pick a colony of bacteria harboring the plasmid DNA of interest into a 12 X
75 mm Falcon tube containing 3 ml of 2xTY media supplemented with the
appropriate antibiotic (typically ampicillin at 100 ug/ml) and incubate at 37deg
C 8-10 hours with shaking at 250 rpm. Transfer the culture
to an Ehrlenmeyer flask containing 50 ml of similar media, and incubate further
for 11-14 hours.

2. Harvest the cells by centrifugation at 3000 rpm for 5 minutes in 50 ml
conical tubes in the Beckman GPR tabletop centrifuge and decant the
supernatant. The cell pellets can be frozen at -70degC at
this point.

5. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal
DNA by pouring through a double-layer of cheesecloth into a new 50 ml conical
tube. Centrifuge at 3,000 rpm for 20 minutes at 4degC in
the Beckman GPR tabletop centrifuge.

6. Decant the supernatant to a 50 ml polypropylene centrifuge tube, add 20 ul
of a 20 mg/ml DNase-free RNase A and incubate in a 37degC
water bath for 30 minutes.

8. Decant the supernatant, resuspend in 7 ml of diatomaceous earth-wash buffer,
and centrifuge as above.

9. Decant the supernatant, resuspend in 7 ml of acetone, and centrifuge as
above.

10. Decant the supernatant and dry in a vacuum oven.

11. Resuspend the pellet in 0.6 ml of 10:1 TE buffer, and elute the bound DNA
by incubation at 65degC for 10 minutes with intermittent
mixing.

12. Remove the diatomaceous earth by centrifugation at 3,000 rpm for 5 minutes
in the in the Beckman GPR tabletop centrifuge.

13. Transfer the supernatant to a 1.5 ml microcentrifuge tube and centrifuge at
12,000 rpm for 5 minutes in a microcentrifuge at room temperature. Transfer
the supernatant to a new 1.5 ml microcentrifuge tube and ethanol precipitate.

The standard method for the miniprep isolation of plasmid DNA includes the
same general strategy as the large scale isolation. However, smaller aliquots
of antibiotic containing liquid media inoculated with plasmid-containing cell
colonies are incubated in a 37degC shaker for 12-16 hours.
After collecting the plasmid containing cells by centrifugation, the cell
pellet is resuspended in a hypotonic sucrose buffer. The cells are
successively incubated with an RNase-lysis buffer, alkaline detergent, and
sodium acetate. The lysate is cleared of precipitated proteins and membranes
by centrifugation, and the plasmid DNA is recovered from the supernatant by
isopropanol precipitation. The DNA is crudely checked for concentration and
purity using agarose gel electrophoresis against known standards. A typical
yield for this method of DNA isolation is 10-15 ug of plasmid DNA from a 6 ml
starting culture.

Since highly supercoiled DNA is desired for double-stranded DNA sequencing, a
modification of this method employing diatomaceous earth (19-22) sometimes is
used for isolation of double-stranded templates for DNA sequencing with
fluorescent primers. After removal of the precipitated proteins and membranes,
the plasmid containing supernatant is incubated with diatomaceous earth and
guanidine hydrochloride and this mixture is added into one of the twenty-four
wells in the BioRad Gene Prep Manifold. The supernatant is removed by vacuum
filtration over a nitrocellulose filter. The DNA-associated diatomaceous earth
is washed to remove the guanidine hydrochloride with an ethanol buffer, and
then dried by filtration. Elution buffer is added to the wells, and the
DNA-containing solution then is separated from the diatomaceous earth particle
by filtration into a collection tube. The collected DNA is concentrated by
ethanol precipitation and crudely assayed for concentration and purity by
agarose gel electrophoresis against known standards. The approximate yield of
double-stranded DNA is 3-5 ug of DNA from 6 ml of starting culture.

Note: This is a typical mini-prep until step 7, where in step 7a you
would precipitate the template and use it for Taq Cycle Sequencing with the
Dye-Labeled Primers, or in step 7b proceed with the diatomaceous earth
purification for Taq Dye-Labeled Terminator Cycle Sequencing Reactions. For
Sequenase Dye-Labeled Terminator Sequencing Reactions use the Midi-prep
procedure detailed above.

5. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal
DNA by centrifugation at 12,000 rpm for 15 minutes in a microcentrifuge at 4deg
C.

6. Transfer the supernatant to a fresh 1.5 ml microcentrifuge tube, incubate in
an ice-water bath for 15 minutes, centrifuge as above for an additional 15
minutes and transfer the supernatant to a clean 1.5 ml tube.

For standard alkaline lysis purification:

7a. Precipitate the DNA by adding 1 ml of 95% ethanol, and resuspend the dried
DNA pellet in 100-200 ul 10:0.1 TE buffer. Electrophorese an aliquot of the
DNA sample on a 0.7% agarose gel to crudely determine the concentration and
purity.

For diatomaceous earth-base purification:

7b. Add 1 ml of de-fined diatomaceous earth in guanidine-HCl (20 mg/ml) and
allow the DNA to bind at room temperature for 5 minutes with occasional mixing.
Meanwhile soak the Prep-A-Gene nitrocellulose membrane in isopropanol for at
least 3 minutes, and assemble the Prep-A-Gene manifold as described in the
manual.

8b. Turn on the vacuum pump and adjust the vacuum level to 8 in. Hg, let the
membrane dry for 1 minute, and then release the vacuum.

9b. Pour the well mixed samples into the wells of the Prep-A-Gene manifold and
filter through at 8 in. Hg until all the liquid is filtered through.

10b. Wash the samples four times with 250 ul of diatomaceous earth-wash buffer,
using a repeat pipette, allowing all of the liquid to filter through between
washes.

11b. Reduce the vacuum to 5 in. Hg before turning the vacuum off at the
stopcock. Without unscrewing the black clamps, release the white clamps and
place the collection rack with clean 1.5 ml screw-capped tubes into the
manifold. Clamp the manifold with the white clamps, and apply 300 ul of 10:1
TE buffer heated to 65degC and pull the eluted DNA through
at 5 in. Hg. After the liquid has filtered through, raise the vacuum to 10-12
in. Hg, and let the membrane dry for 1 minute.

12b. Turn off the vacuum at the stopcock and remove the collection rack
containing the tubes. Ethanol precipitate the DNA and resuspend the dried DNA
pellet in 30 ul of 10:0.1 TE buffer.

Double-stranded M13RF is isolated for use in M13 SmaI cut,
dephosphorylated vector preparation, described below. The growth conditions of
M13-infected bacterial cells (see Figure 1) appears convoluted, but result in a
maximal amount of M13 RF molecules per cell. After the M13RF containing
bacterial cells are harvested by centrifugation, the double-stranded molecules
are isolated using the cesium chloride method for large scale plasmid
isolation, as described above. This briefly entailed alkaline cell lysis,
sodium acetate precipitation of detergent solubilized proteins and membranes,
polyethylene glycol DNA precipitation, and extraction of ethidium bromide
stained DNA from a cesium chloride gradient after ultracentrifugation. After
removal of the ethidium bromide on an ion-exchange column, the DNA containing
fractions are detected by A260 measurement and pooled, and the DNA is
concentrated by ethanol precipitation and assayed by restriction enzyme
digestion and agarose gel electrophoresis.

Protocol

1. Prepare an early log phase culture of JM101 by inoculating an Ehrlenmeyer
flask containing 50 ml of 2xTY with a glycerol stock of JM101 and
pre-incubating for 1 hour in a 37degC water bath, with no
shaking. Pick a plaque representing the desired M13 clone into four 1.5 ml
aliquot of early log phase JM101, and incubate according to the procedure
displayed in Figure 1 to result in 4 liters of M13-infected bacteria.

2. Harvest the cells by centrifugation at 7000 rpm for 20 minutes in 500 ml
bottles in the RC5-B using the GS3 rotor. Resuspend the cell pellets in fresh
2xTY media to remove contaminating extracellular phage and transfer to two
bottles, centrifuge as before, and decant the media. The cell pellets can be
frozen at -70degC at this point.

3. Resuspend the cell pellets in a total of 120 ml (30 ml for each bottle) of
1X STB buffer by gently teasing the pellet with a spatula. Add a total 24 ml
of lysozyme solution (6 ml for each bottle), gently mix, and incubate for 5
minutes in an ice-water bath.

4. Add 48 ml of 50:2:10 TTE buffer (12 ml for each bottle) and 2 ml of RNase A
(10 mg/ml) (0.5 ml for each bottle), gently mix, and incubate in an ice-water
bath for 5 minutes.

5. Clear the lysate of precipitated SDS, proteins, membranes, and chromosomal
DNA by pouring through a double-layer of cheesecloth. Transfer the lysate into
250 ml centrifuge bottle, centrifuge at 10,000 rpm for 30 minutes at 4deg
C in the RC5-B using the GSA rotor.

6. Add 6 ml of 5 mg/ml ethidium bromide, and cesium chloride such that the
final concentration of cesium chloride is 1 g/ml.

7. Transfer the sample into 35 ml polyallomer centrifuge tubes and top off with
a 1:1 solution of 100:10 TE buffer and cesium chloride, remove air bubbles,
seal with rubber stoppers, and crimp properly.

8. Centrifuge at 60,000 rpm to 16-20 hours at 15-20degC in
the Sorvall OTD-75B ultracentrifuge using the T-865 rotor.

9. Visualize the ethidium bromide stained DNA under long-wave UV light, and
remove the lower DNA band using a 5 cc syringe and a 25 gauge needle. (It may
be helpful to remove and discard the upper band first).

11. Pool fractions with an A260 of 1.00 or greater into 35 ml Corex glass
tubes, add one volume of ddH2O, and ethanol precipitate by adding 2.5 volumes
of cold 95% ethanol. Incubate at least 2 hours at -20degC,
centrifuge at 10,000 rpm for 45 minutes in the RC5-B using the SS-34 rotor.
Gently decant the supernatant, add 80% ethanol, centrifuge as before, decant,
and dry the DNA pellet in a vacuum oven.

This isolation procedure (23) is the method of choice for preparation of
M13-based templates to be used in Sequenase[TM] catalyzed
dye-terminator reactions. A pre-incubated early log phase JM101 culture is
prepared by transferring a thawed glycerol stock into 50 ml of liquid media and
incubating for 1 hour at 37degC with no shaking. M13
plaques are picked with a sterile toothpick and placed into 1.5 ml aliquots of
the early log phase JM101 culture, which are incubated in a 37deg
C shaker for 4-6 hours. After incubation, the bacterial
cells are pelleted by centrifugation and the viral containing supernatant is
transferred to a clean tube. The phage particle are precipitated with PEG,
collected by centrifugation, and the pellet is resuspended in buffer. The
phage protein coat is denatured and removed by one phenol and two ether
extractions. After ethanol precipitation, the dried DNA pellet is resuspended
in buffer, and the concentration and purity crudely are assessed by agarose gel
electrophoresis against known standards.

Protocol

1. Prepare an early log phase culture of JM101, as above, and pick M13-based
plaques with sterile toothpicks into 12 X 75 mm Falcon tubes containing 1.5 ml
aliquots of the cells. Incubate for 4-6 hours at 37degC
with shaking at 250 rpm.

2. Transfer the culture to 1.5 ml microcentrifuge tubes and centrifuge for 15
minutes at 12,000 rpm at 4degC.

3. Pipette the top 1 ml of supernatant to a fresh 1.5 ml microcentrifuge tube
containing 0.2 ml 20% PEG/2.5 M NaCl to precipitate the phage particles. Mix
by inverting several times and incubate for 15-30 minutes at room
temperature.

4. Centrifuge for 15 minutes at 12,000 rpm at 4degC to
collect the precipitated phage. Decant the supernatant and remove residual PEG
supernatant by suctioning twice.

This semi-automated method is a modification of a previously reported
procedure (24,25), and allowed the simultaneous isolation of 48 single-stranded
DNAs per Biomek 1000 robotic workstation within 3 hours (26). Basically, M13
plaques are picked with sterile toothpicks into aliquots of early log phase
JM101, prepared as discussed above. The phage infected cultures are incubated
in a 37degC shaker for 4-6 hours, transferred into
microcentrifuge tubes, centrifuged to separate bacterial cells from the viral
supernatant, and then carefully placed on the Biomek tablet. For each sample,
two 250 ul aliquots are robotically distributed into two wells of a 96-well
microtiter plate, and this process is repeated for each of the 48 samples until
the entire 96 wells are filled. A solution of polyethylene glycol (PEG) then
is added robotically to each well and mixed. The microtiter plate is covered
with an acetate plate sealer, incubated at room temperature to precipitated the
phage particles, and then centrifuged. The supernatant then is removed by
inverting the plate and gently draining on a paper towel, without dislodging
the pellet. After placing the microtiter plate back on the Biomek, a more
dilute PEG solution is robotically added to each well. The plate then is
covered with another sealer and centrifuged again. This rinse step aided in
the removal of contaminating proteins and RNA. After removing the supernatant,
as before, and placing the microtiter plate back on the Biomek, a Triton X-100
detergent solution is robotically added to each well. The plate is agitated
gently and the sample from each pair of wells is robotically transferred to
microcentrifuge tubes, which then are capped and placed in an 80deg
C water bath for 10 minutes to aid in the detergent
solubilization of phage coat proteins. After a brief centrifugation to collect
condensation, the single-stranded DNA is ethanol precipitated, dried, and
resuspended. An aliquot from each DNA sample is subjected to agarose gel
electrophoresis to crudely assay concentration and purity. The yield of
single-stranded template is approximately 2-3 ug per sample.

Protocol

The entire procedure will require 9 rows of P250 tips (counting from the
center of the Biomek tablet towards the left) for the isolation of 48 templates
(48ISOL). The reagent module should contain PEG-2000, Triton-Tris-EDTA, and
ethanol-acetate, respectively.

1. Prepare an early log phase JM101 culture in 50 ml of 2xTY, as above.

3. Separate the bacterial cells from the viral-containing supernatant by
centrifugation at 12,000 rpm for 15 minutes at 4degC.
Carefully open the tubes and place on the Biomek tablet..

4. The Biomek will distribute two 250 ul aliquots of viral supernatant per
sample into the wells of a 96-well flat-bottomed microtiter plate (Dynatech).
The Biomek then will add 50 ul of 20% PEG/2.5 M NaCl solution to each well, and
mix by pipetting up and down.

5. Cover the plate with an acetate plate sealer and incubate at room
temperature for 15 minutes.

6. Pellet the precipitated phage by centrifuging the plate at 2400 rpm for 20
minutes in a Beckman GPR tabletop centrifuge. Remove the plate sealer and
drain the PEG from the plate by gently draining upside down on a Kimwipe.

7. Return the plate to the tablet, and the Biomek will robotically add 200 ul
of PEG:TE rinse solution to each well. Cover the plate with a plate sealer,
centrifuge, and drain, as above.

8. Return the plate to the tablet, and the Biomek will add 70 ul of TTE
solution to each well. Remove and gently agitate to resuspend.

9. The Biomek then will robotically pool the contents from each pair of wells
into 1.5 ml microcentrifuge tubes.

10. Incubate the tubes at 80degC for 10 minutes to denature
the viral protein coat and then centrifuge briefly to reclaim condensation.

11. Ethanol precipitate the DNA by adding 500 ul ethanol/acetate to each tube,
as described above.

A manual as well as an automated procedure is given below. The automated
method is a modification of a previously reported procedure (4) which allows
simultaneous isolation of 96 double stranded DNAs per Biomek 1000 Automated
Laboratory Workstation within two hours. Basically colonies containing
double-stranded plasmids are picked with sterile toothpicks into media and
incubated at 37degC for 24 hours with shaking at 350 rpm. These cells are
harvested by centrifugation and the pellets are either manually or robotically
resuspended by the addition of TE-RNase solution. An alkaline lysis solution
is used to lyse the cells and the lysate is precipitated with KOAc. The lysate
is cleared by filtration and further concentrated by ethanol precipitation.
An aliquot from each DNA sample is subjected to agarose gel eletrophoresis to
crudely assay concentration and purity. The yield of double stranded template
is approximately 3 mg per sample.

Protocol

Manual Double stranded isolation method

The following is a manual, 96 well, double stranded sequencing template isolation
procedure that has been developed in our laboratory. A similar procedure that
has been automated on the Biomek is presented elsewhere herein.

1. Pick individual shotgun clones off a plate with a steril tooth pick and
deposit each separately into 96 well block containing 1.75 ml of TB media per
well. Keep toothpick in media for about 5 minutes to allow the cells to defuse
into the media, remove the toothpicks, cover the 96 well block with the loose
fitting lid, and allow the cells to grow for 24 hours in the 37degC shaker/incubator
at 350 rpm.

2. Remove block from the shaker/incubator and collect the cells by centrifugation
at 2500 rpm for 7 minutes. The cells can be stored frozen at -20degC in the block
at this stage.

3. After thawing the cells, add 100ul TE-RNAse-A solution containing RNAse T1,
mix by pipetting up and down 4-5 times to resuspend the cell pellet and then
incubate in the 37degC incubator/shaker for 5 minutes at 350 rpm to mix more thoroughly.

4. Remove the block from the incubator/shaker and then add 100ul of alkaline
lysis solution. Shake the block by hand to mix the reagents and then incubate
at room temperature for 1 hour with intermittent swirling.
5. Then add 100ul of either 3M potassium or sodium acetate, pH 5, and place
the block in the 37degC shaker/incubator for 5 minutes at 350 rpm to thoroughly mix
and shear genomic DNA to reduce the viscosity of the solution. Place the block at
-20degC for 30 minutes.

6. Centrifuge the block in the GPR centrifuge at 3000 rpm at 4degC for 30 minutes.

7. Carefully remove 200 ul of the supernatant from each well in the 96 well block
with the 12 channel pipetter and transfer them to a v-bottom microtiter plate, being
careful not to transfer any cell debris.

8. Transfer 10 ul of supernatant into the respective cycle sequencing reaction tubes, and precipitate with 150 ul of 95% ethanol
(without added acetate). After storage at -20degC for 30 minutes, the pellet is
collected by centrifugation, washed three times with 70% ethanol, and dried directly
in the cycle sequencing reaction tubes.

9. Prior to adding the fluorscent terminator
cycle sequencing reaction mix, the dried templates should be stored at -20degC. An
additional 75 ul of the supernatant is transfered to a Robbins PCR reaction tube
(in 96 well tube format) and precipitated with 200 ul of 95% ethanol, washed three
times with 70% ethanol, and stored dry at -20degC for future use.

The following is an automated, 96 well, double stranded sequencing template isolation
procedure that has been developed in our laboratory.

1. Pick colonies using a toothpick into 1.8 ml TB with TB salts containing appropriate
antibiotic and shake for 22-24 hours at 350 rpm in a 96 well block with cover.

2. Harvest cells by centrifugation at 1800 rpm for 7 min. Pour off supernatant and
allow pellets to drain inverted. Cell pellets may be frozen at this point if necessary.

3. Turn on Biomek, begin the program DSISOL2 and set up the Biomek as indicated in
the configuration function on the screen. Specifically, you should put TE-RNase solution
in the first module, alkaline lysis solution in the second reagent module and 3 M KOAc,
pH 4.8 in the third module.

4. Place the 96 well block containing cells onto the Biomek tablet at the position
labeled "1.0 ml Minitubes". Place a Millipore filter plate in the position labeled
"96well flat bottomed microtitre plate".

5. Press ENTER to continue with the program.

6. First the Biomek will add 100 ml TE-RNase solution to the cell pellets and mix to
partially resuspend.

7. Next, the biomek will add 100 ml alkaline lysis solution to the wells of the
filter plate.

8. The biomek then will mix the cell suspension again, transfer the entire volume
to the filter plate containing alkaline lysis solution, and mix again. Set up the
filtration apparatus with a clean 96 well block to collect the filtrate (wash and
reuse the block used for growth).

9. The biomek will add 100 ml 3M KOAc, pH 4.8 to the wells of the filter plate and
mix at the sides of the wells. Some choose to place the filter plate at -20degC for
5 minutes at this point. Transfer the filter plate to the QiaVac Vacuum Manifold 96
and filter using water vacuum only (do not do a harsh filtration as the plates are
fragile and will loose their seal). This will typically take less than 20 minutes.

10. The supernatant collected in the 96 well block is the crude DNA and must be ethanol
precipitated before use by the addition of 1 ml 100% ethanol and incubation at -20degC
for at least 30 minutes.

Genomic DNA isolation is performed according to the FBI protocol (27).
After the blood samples (stored at -70degC in EDTA
vacutainer tubes ) are thawed, standard citrate buffer is added, mixed, and the
tubes are centrifuged. The top portion of the supernatant is discarded and
additional buffer is added, mixed, and again the tube is centrifuged. After
the supernatant is discarded, the pellet is resuspended in a solution of SDS
detergent and proteinase K, and the mixture is incubated at 55deg
C for one hour. The sample then is phenol extracted once
with a phenol/chloroform/isoamyl alcohol solution, and after centrifugation the
aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol
precipitated, resuspended in buffer, and then ethanol precipitated a second
time. Once the pellet is dried, buffer is added and the DNA is resuspended by
incubation at 55degC overnight, the genomic DNA solution is
assayed by the polymerase chain reaction.